Regulated trafficking and targeting of membrane proteins to specific subcellular domains is an essential aspect of the organization of very large cells such as neurons and muscle fibers. The goal of this project is to understand how subcellular domains are organized in these cells during differentiation, and how they are subsequently shaped by cellular activity. We believe that the formation of such domains depends on changes in the organization of the Golgi complex, the strategic cellular center for membrane protein sorting and targeting. During muscle differentiation and maturation, the Golgi complex undergoes striking changes. Neither their mechanism nor their regulation is understood. The mouse muscle cell line C2 is our model to study differentiation. During differentiation, the Golgi complex appears to fragment into small stacks of cisternae which are positioned along the outer nuclear membrane of the myotube nuclei and in rows in the cytoplasm. We have demonstrated that the Golgi complex of myotubes is made of independent elements, which are localized at the endoplasmic reticulum (ER) exit sites. The process which underlies the changes of the Golgi complex during differentiation resembles that taking place when microtubules are disrupted in all mammalian cells. We have proposed that changes in the nucleation of microtubules and in their polarity relative to the nuclear membrane explain the Golgi complex fragmentation in muscle. These results are important because they show that simple drug treatments, for example with the microtubule-depolymerizing drug nocodazole, mimick and can serve as a model for the physiological events taking place during muscle differentiation. In the past year we have focused on the ER exit sites. These have been thought to be static and probably uniformly distributed along the ER membranes. Our results however show that ER exit sites are reorganized during differentiation and are also reorganized following treatments by drugs such as nocodazole and brefeldin A. Interestingly, it appears that this is not merely reflecting a reorganization of the whole ER but that the ER exit site localization is directly controlled. Several experiments are in progress to investigate how such control takes place. We are transfecting C2 cells to obtain simultaneous expression of two fluorescent proteins, for example one labeling the ER and the other one the ER exit sites. With a combination of two variants of the green fluorescent protein GFP we are then able to visualize both markers simultaneously in live cells, a powerful tool. We are assessing the effect of various drugs on the ER exit sites by EM immunogold, while micro-injection of antibodies and dominant negative constructs will allow us to learn more about the mechanism of ER exit site localization and how it changes during differentiation. We have also continued to investigate how the Golgi complex is organized in muscle fibers in vivo. In order to test the hypothesis that intermediate filaments may contribute to the localization of the Golgi complex, we started to examine the distribution of various markers in muscles from a desmin -/- knockout. A preliminary examination revealed no gross defects in the localization of the Golgi complex. Interestingly, microtubules appeared upregulated. The examination of transgenic mice should be useful to determine the role of specific molecules in the cellular organization of muscle fibers in health and disease.